title: HPLC profiling with at-line microdilution assay for the early identification of anti-fungal compounds in plants from French Polynesia.
abstract: BACKGROUND
  The search for anti-fungal compounds has maintained a scientific interest notably due to existing difficulties in the treatment of mycoses and their increasing occurrence in hospitals.
  
  
  OBJECTIVE
  Development of a simple method to rapidly identify anti-fungal compounds in crude plant extracts based on a HPLC microfractionation approach combined with an at-line anti-Candida assay.
  
  
  METHODS
  The scale of the semi-preparative HPLC microfractionation was adapted to fit the sensitivity of the Candida albicans anti-fungal in a 96-well microdilution assay. This format is also compatible for MS and NMR dereplication of the active compounds.
  
  
  RESULTS
  Based on the screening of 12 crude extracts of plants from French Polynesia, three plants, which displayed various levels of anti-fungal activities, were selected to assess the efficiency of the HPLC anti-fungal profiling and the scale necessary for microfractionation. The same anti-Candida assay was performed on the HPLC microfractions collected using a generic profiling method. Analysis of active microfractions by MS and NMR issued from the most active extract enabled an efficient dereplication of the compounds responsible for the anti-fungal activity.
  
  
  CONCLUSIONS
  A generic HPLC anti-fungal profiling method was developed which revealed that only 50 mg of crude extract were sufficient for a rapid identification of compound(s) responsible for the anti-Candida activity. This approach was illustrated by the study of Alphitonia zizyphoides, a plant traditionally used to treat dermatomycoses.
authors.affiliation: School of Pharmaceutical Sciences, EPGL, University of Geneva, University of Lausanne, Quai Ernest-Ansermet 30, CH-1211, Geneva 4, Switzerland.
authors.forename: Samuel
authors.initials: S
authors.lastname: Bertrand
authors.forename: Charlotte
authors.initials: C
authors.lastname: Petit
authors.forename: Laurence
authors.initials: L
authors.lastname: Marcourt
authors.forename: Raimana
authors.initials: R
authors.lastname: Ho
authors.forename: Katia
authors.initials: K
authors.lastname: Gindro
authors.forename: Michel
authors.initials: M
authors.lastname: Monod
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Wolfender
mesh_terms: D000935:Antifungal Agents / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D002176:Candida albicans / Q000187:drug effects*; D005591:Chemical Fractionation / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D008826:Microbial Sensitivity Tests; D010936:Plant Extracts / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D011114:Polynesia; D027823:Rhamnaceae / Q000737:chemistry*; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D000935:Antifungal Agents; D010936:Plant Extracts
keywords: Alphitonia zizyphoides; Candida albicans; anti-fungal; betulinic acid; microfractionation